Conjugated estrogens obtained from pregnant mares' urine are a mixture of the sodium salts of 3-sulfated estrogenic substances. These 3-sulfate estrogens are secreted in substantial quantities in the urine of pregnant mares (pregnant mares' urine; PMU). Following this discovery in the 1930's, a pharmaceutical preparation of estrogens obtained from pregnant mares was marketed as a hormone replacement therapy in 1942, as PREMARIN®. PREMARIN® is still on the market today as an important therapeutic agent.
The major estrogenic substances in PREMARIN® are sodium estrone sulfate and sodium equilin sulfate. Also present are sodium sulfate conjugates of 17α-dihydroequilin, 17α-estradiol, and 17β-dihydroequilin. Tablets for oral administration are available in 0.3 mg, 0.45 mg, 0.625 mg, 0.9 mg, and 1.25 mg strengths of conjugated estrogens. PREMARIN® is also available as a cream.
Pharmaceutical preparations of conjugated estrogens are used to treat symptoms of menopause and other conditions as a form of hormone replacement therapy. Menopause is generally defined as the last natural menstrual period of a woman and is characterized by the cessation of ovarian function, leading to the substantial diminution of circulating estrogen in the bloodstream. Menopause is usually identified, in retrospect, after 12 months of amenorrhea. It is not a sudden event, but is often preceded by a time of irregular menstrual cycles prior to eventual cessation of menses. Following the cessation of menstruation the decline in endogenous estrogen concentrations is typically rapid. There is a decrease in serum estrogens from circulating levels ranging from 40-250 pg/mL of estradiol and 40-170 pg/ml of estrone during ovulatory cycles to less than 15 pg/mL of estradiol and 30 pg/mL of estrone in postmenopausal women.
As these estrogens decline during the time preceding (perimenopuase) and following the menopause (postmenopause), various physiological changes may result, including vulvar and vaginal atrophy causing vaginal dryness, pruritus and dysparenuia, and vasomotor instability manifested as hot flushes. Other menopausal disturbance may include depression, insomnia, and nervousness. The long-term physiologic effects of postmenopausal estrogen deprivation may result in significant morbidity and mortality due to increase in the risk factors for cardiovascular disease and osteoporosis. Menopausal changes in blood lipid levels, a major component of the pathogenesis of coronary heart disease (CHD), may be precursors to increased incidence of ischemic heart disease, atherosclerosis, and other cardiovascular disease. A rapid decrease in bone mass of both cortical (spine) and trabecular (hip) bone can be seen immediately after the menopause, with a total bone mass loss of 1% to 5% per year, continuing for 10 to 15 years.
Hormone replacement therapy (HRT) using estrogens is beneficial for symptomatic relief of hot flushes and genital atrophy associated menopause and is useful for prevention of postmenopausal osteoporosis. HRT has been recognized as an advantageous treatment for relief of vasomotor symptoms. There is no acceptable alternative to estrogen treatment for the atrophic changes in the vagina; estrogen therapy increases the vaginal mucosa and decreases vaginal dryness. Long term HRT is also used to prevent osteoporosis because estrogen therapy can decrease bone loss, reduce spine and hip fracture, and prevent loss of height. In addition, HRT has been shown to be effective in increasing high density Lipoprotein-cholesterol (HDL-C) and in reducing low density lipoprotein cholesterol (LDL-C), affording possible protection against CHD. HRT also can provide antioxidant protection against free radical mediated disorders or disease states. Estrogens have also been reported to confer neuroprotection, and inhibit neurodegenerative disorders, such as Alzheimer's disease. PREMARIN® is a popular HRT available worldwide, and is believed to be an effective treatment for all of these disorders.
Natural mixtures of conjugated estrogens such as found in the urine of pregnant mares have proved particularly effective and well tolerated for HRT. The dissolved solids content in PMU may naturally vary within wide ranges, and may generally lie in a range of 40-90 g dry matter per liter. In addition to urea and other usual urine contents, phenolic constituents are contained in the solids content of the PMU in quantities of about 2%-5% by weight related to dry matter. These phenolic constituents include cresols and dihydro-3,4-bis[3-hydroxyphenyl)methyl]-2(3H)-furanone (HPMF). These phenolics may be present in free or conjugated form. The PMU contains a natural mixture of estrogens which is largely present in conjugated form, e.g. as sulfuric acid semi-ester sodium salt (referred to hereinafter as “sulfate salt”). The content of conjugated estrogens (calculated as estrogen sulfate salt) may be between 0.3% and 1% by weight relative to dry matter.
Conventionally, conjugated estrogens are obtained from PMU by various drying, trituration, and liquid-liquid extraction methods. Conjugated estrogens have been obtained by extraction with a polar organic solvent which is immiscible with water, or only slightly miscible, such as ethyl acetate, n-butanol or cyclohexanol. In such liquid-liquid extractions, however, a number of problems occur, such as foaming, sedimentation, emulsification and poor phase separation. Generally several extraction steps are required, which results in losses and only partial recovery of the estrogen content. Conjugated estrogens have also been isolated by extracting PMU and an organic solvent such as n-butanol or instead by adsorption on charcoal. Such methods have involved a multiplicity of individual process operations, involving back extraction and repeated transfer between n-butanol and aqueous solutions. Such repeated extractions generally result in losses of conjugated estrogens and thus only partial recovery of the estrogen content of the PMU. Examples of extraction process are disclosed in U.S. Pat. Nos. 2,696,265; 2,711,988 and 2,834,712.
Several newer purification methods have been developed, for example employing non-ionic resin adsorption, for example as disclosed in U.S. Pat. No. 7,964,586 and U.S. Pat. No. 5,723,454, and ion exchange resins, for example as disclosed in U.S. Pat. No. 6,855,704 and U.S. Pat. No. 7,081,451.
The isolation process for obtaining purified conjugated estrogens from PMU must preserve the estrogenic sulfate group and remove non-sulfated estrogenic components. Prior art processes for the purification result in solids suitable for pharmaceutical use containing about 25% of conjugated estrogens. The exact quantity of conjugated estrogens must be assayed, since the quantity of conjugated estrogens in pharmaceutical preparations is based on the actual assay.
Furthermore, non-sulfated “non-conjugated” components are undesirable impurities in conjugated estrogen mixtures. The USP monograph for conjugated estrogens specifies that the limit of free steroid impurities should not exceed 1.3%.